The proposed research seeks to define completely the requirements for growth and differentiation of several types of cultured human diploid cells. During the coming year, emphasis will be given to gaining a more complete understanding of the growth requirements of fibroblasts from skin and fetal lung and of keratinocytes. These cell types can already be grown with minimal amounts of serum protein. In addition, for the fetal lung fibroblasts, DNA synthesis equivalent to one half that obtained with serum protein can be obtained in the complete absence of serum protein through the use of a mixture of hormones in conjunction with an optimized medium, polylysine-coated culture vessels, and low temperature trypsinization. Major emphasis will be given to lipid requirements and to further refinement of the hormonal mixture during the coming year, together with continuing attempts to isolate from serum or other sources those growth-promoting activities that cannot be fully replaced by lipids or hormones. The studies with lipids and hormones will be extended to keratinocytes. Preliminary studies involving optimization of the culture medium and conditions will also be initiated with human chondrocytes and mammary epithelial cells. These will be followed by hormonal and lipid studies as rapidly as the preliminary phases can be completed. The media and cell culture technology that will be developed during this research program will greatly facilitate future research in many different areas of human cell biology and biomedical science.